1. Field of the Invention
The present invention relates to a method for expressing a gene product that could not be expressed because of blockage occurring in a regulatory region of the gene. The present invention also relates to a method of restoring iodide transport in cells defective in iodide transport. The present invention is also directed to a method of treating tumors by expressing tumor specific therapeutic response element in a cancerous cell in which the response element was blocked from expression.
2. Brief Description of the Related Art
The initial step in the synthesis of thyroid hormone is the active transport of iodide, mediated by the sodium-iodide symporter (NIS) located in the basolateral membrane of thyroid follicular cells (1). This iodide-concentrating ability of thyroid follicular cells is exploited for the treatment of differentiated thyroid epithelial carcinomas, using therapeutic dosages of I-131. Loss of iodide concentrating ability, in the face of distantly metastatic disease, results in significant morbidity and mortality for around 10% of patients with differentiated thyroid cancers (2). In addition, anaplastic thyroid cancers, which are unable to take up radioactive iodide and do not respond to systemic chemotherapies, are invariably fatal.
The complementary DNA sequence for human NIS (hNIS), as well as the exon-intron organization, have been revealed by Smanik et al (3,4). The cloning and characterization of a 1.3 kb region of the upstream regulatory region was reported and a minimal essential hNIS promoter that shows tissue-specific expression in a human thyroid cell line was defined (5). Other investigators have further evaluated hNIS promoter constructs (6,7). It is possible that alterations in hNIS expression, responsible for loss of iodide-concentrating ability in human thyroid cancer metastases, may correspond to changes in hNIS promoter activity. This may be similar to the loss of E-cadherin expression demonstrated in human thyroid cancer cell lines, correlating to methylation of CpG islands in the E-cadherin promoter (8). Since the hNIS promoter has CpG-rich regions, as well as additional CpG islands downstream from the transcription start site, DNA methylation may be responsible for alterations in hNIS expression. The DNA sequence corresponding to the minimal essential promoter of the gene encoding hNIS has been reported (49). The contents of this reference are incorporated herein by reference in their entirety.
Nearly half of all human genes have CpG islands associated with transcriptional start sites. Unmethylated CpG islands are seen in highly transcribed genes, while heavily methylated CpG islands inhibit transcription (9). Although overall DNA methylation is often decreased in cancers, CpG islands in critical gene promoter regions can become hypermethylated, resulting in loss of gene expression (10). Such methylation may be effective in silencing gene expression despite variable degrees of CpG site methylation from 20 to 100% (11). Laboratory and clinical studies have suggested that chemical agents may demethylate these regions and restore gene expression. Examples include use of: 5-azacytidine to restore expression of O6-methylguanine-DNA methyltransferase in human cervical, brain, and colon carcinomas (12,13); phenylacetate to induce fetal hemoglobin expression in human leukemic cells (14); and sodium butyrate to induce prolactin receptor expression in human breast cancer cells (15).
The present invention is directed to a method of controlling transcriptional expression of a gene by a multi-faceted epigenetic approach. That is, the gene of interest is transcriptionally regulated by modifying the chromosome structure without mutating the bases in the DNA. Several examples of epigenetic modifications of the chromosome are included in the invention, without limitation. Administration of butyrate, for example, results in the acetylation of histones, thereby resulting in a more “open” chromatin structure facilitating transcriptional activation (50–52). Phosphorylation of the nucleosome similarly alters the chemical and physical properties of the nucleosome, thereby allowing greater or lesser access to a specific trans- acting factor that may bind to a specific region on the DNA (55–56). Also, administration of certain critical amounts of endogenous or exogenous DNA binding agents, such as polyamines, cause the level of transcriptional activity of a gene to alter in part because of the change in the chemical and physical properties of the modified nucleosome (53–54). Thus, it can be seen that the methods of demethylating or inhibiting methylation of DNA, as exemplified herein, serves as merely an illustration of transcriptional regulation engendered by epigenetic modification of chromosomes.
In the present invention methylation of the characterized hNIS promoter, and potentially regulatory downstream regions, and their correlation with loss of hNIS mRNA expression, as well as clinical loss of iodide uptake, in samples of thyroid tumor tissues were tested. In addition, using human thyroid carcinoma cell lines and putative demethylation agents, the reversibility of loss of hNIS mRNA expression and functional activity, measured as iodide uptake were evaluated.